Resistance of tumors to chemotherapy and radiotherapy may be attributed in part to the unique ultrastructure of the tumor neovasculature and physiological microenvironment of tumor cells. Tumor neovasculature is a disordered network of blood vessels containing multiple anastomotic branches and shunts, resulting in spatial and temporal heterogeneity in blood perfusion. This causes portions of the tumor to show hypoxic oscillations or become frankly hypoxic with pO2 levels ranging from 1-10%. Hypoxia contributes to treatment resistance in a multi-factorial manner. For example, reduction in proliferation and increases in drug resistance pathways, such as glutathione synthetase, occur. In the case of radiation therapy, the presence of molecular oxygen is necessary for the most effective forms of DNA damage. Irregularities in vascular transport also result in inefficient drug delivery. In such a milieu, the effectiveness of chemo- and radiotherapy are unpredictable and ineffective. In the presence of low vascular pO2 typical of tumor microvasculature, erythrocytes become slightly crenated and less deformable, leading to rouxleux formation and increased flow resistance. Erythrocytes from patients with sickle cell anemia undergo more substantial changes under low pO2 conditions. SS hemoglobin desaturates in a time-dependent manner leading to polymerization of deoxy-HbS(α2βs2) tetramers and formation of rigid spicules that attach to the cytoskeleton. A population of nondeformable sickled erythrocytes (SRBCs) emerges that tends to attach to and occlude microvessels. Moreover, activated SS RBCs display multiple adhesion receptors such as BCAM/Lu, ICAM-4 and α4β1, which adhere abnormally to cognate endothelial ligands laminin-α5, ανβ, and VCAM-1, respectively, and are thought to play a major role in SS cell-mediated vaso-occlusive events. Several of these cognate endothelial ligands such as laminin-β5, ανβ3, and VCAM-1 are up-regulated on sickle cell vasculature by oxygenation/reperfusion. Notably, they are also broadly expressed and upregulated on tumor neovessels as a consequence of angiogenesis and tumor hypoxia. Thus, SS cells possess properties that are well suited for adherence to, entrapment in and occlusion of tumor neovessels. Furthermore, SS RBCs involved in vaso-occlusive events produce superoxide/peroxide-driven hydroxyl radicals leading to membrane peroxidation and hemolysis. Hemoglobin released from hemolyzed SS cells is rapidly oxidized from ferro- to ferri-hemoglobin (methemoglobin) generating highly lipophilic heme-nitrosyl complexes that readily intercalate into cell membranes. Intracellular heme and its oxidative product free iron are highly toxic to cells especially in the presence of heme oxygenase inhibitors. Heme catalyzes the oxidation of membrane lipids and DNA and activates caspases and cathepsins leading to perturbations of cytoskeleton and apoptosis (40, 41).
Heme oxygenase (HO-1), a 32-kD microsomal membrane enzyme, is overexpressed in many tumor cells. It is activated by heme, HIF-1 (hypoxia inducible factor-1) and other stress-associated proteins (42-44). It protects cells from oxidative stress by detoxifying ferric ion and cleaving β-type heme molecules to form equimolar quantities of antioxidant bilirubin and antiapoptotic carbon monoxide. HO-1 is upregulated in transgenic sickle mice by hemin and its inhibition by tin protoporphyrin exacerbates vascular stasis and vaso-occlusion (45). Pharmacological inhibition of HO-1 in vivo also enhances the prooxidant and proapototic consequences of heme product exposure (46-48). We infused rhodamine-labeled SS RBCs and normal RBCs into mice bearing skin-fold window chambers containing 4T1 mammary carcinomas. At this time a robust neovasculature was observed. We monitored SS RBC behavior via intravital microscopy. After demonstrating occlusion of tumor neovessels, we examined the ability of SS cells to induce a tumoricidal response against an established carcinoma in the presence of a heme oxygenase inhibitor. We surmised that in the absence of the cytoprotective effect of heme oxygenase, constitutive heme and related oxidative products released from SS cells in occluded tumor microvessels would be free to induce a significant anti-tumor response. Attempts to target tumors with liposomes and nanoparticles have yet to surmount the problems of disintegration in the bloodstream, uptake by macrophages and Kupffer cells in liver and spleen and inablilty to pass through the endothelial cell barrier. Pegylated liposomes show reduced uptake by macrophages and a prolonged half-life but still have not exhibited sufficient localization to tumor tissues. Stealth liposomes in which a targeting molecule is attached to a pegylated residue have shown localization to tumors in vivo but to date, no significant therapeutic effects. Once localized to the target cells, liposomes must then traverse the cell membrane. Fusigenic molecules to promote fusion with the cell membrane, penetratin and TAT-mediated translocation, receptor mediated endocytosis have been employed to address this problem but to date have produced no convincing anti-tumor effects (Lasic DD Applications of Liposomes in Handbook of Biological Physics, vol. 1, edited by R Lipowsky & E Sackmann, Elsevier Science, p. 491-519 (1995))
The instant inventors also recognized that unlike monoclonal antibodies, these very same SS erythrocytes, their nucleated precursors, sickle hemoglobin variants, erythroleukemia cells are carriers of potent tumoricidal agents into tumors. The inventors contemplate that hemoglobin in nucleated SS precursor erythroblasts is equally effective at polymerizing under hypoxemic conditions while nucleation endows them with the ability to be transduced by oncolytic viruses and to carry these viruses specfically into tumor tissue. Indeed, by placing these viruses under control of a hypoxia responsive transcriptional control element (HRE), they are activated selectively in the hypoxemic tumor vasculature rather than normoxemic tissues (Table 1). The oxygen tension of tumors (as opposed to normal tissues) is in a range appropriate for activation of the HRE especially in concatenated and polymerized form. The nucleated sickle erythroblasts and activated erythroleukemia cells of the present invention are a major improvement over monoclonal antibody, liposome and nanoparticle technology since: (i) they exhibit a higher degree of tumor localization, (ii) they penetrate the tumor vasculature more effectively and obstruct or occlude tumor microvessels, (iii) they can be transduced with tumor specific oncolytic viruses such as the self-replicating, RNA alphaviruses and adenoviruses or vectors comprising tumor specific tumoricidal transgenes. Tumor specific oncolytic-oncotropic viruses proliferate in and lyse the SS or erythroleukemia cells and then proceed to infect and kill tumor cells specifically via a bystander effect. Because of the bystander effect and the specificity of oncolytic viruses for tumor cells only a few tumor cells need be infected by the virus in order to initiate a generalized tumoricidal response. (iv) by placing the tumor specific oncolytic viruses under control of concatenated hypoxia-responsive elements, the activation of the tumor specific oncolytic viruses occurs selectively in the hypoxemic microenvironment of the tumor.
The present invention provides a remedy for specificity and efficacy. It uses a natural cell, the erythrocyte of sickle cell anemia, its nucleated precursors and sickle hemoglobin variants, erythroleukemia cells which the inventors have observed to have a proclivity to deposit selectively in the tortuous neovasculature of tumors. Indeed, sickled cells show exquisite specificity for tumor microvasculature. In the hypoxemic environment of tumors, SS hemoglobin polymerizes resulting in an increase in membrane rigidity, upregulation of adherence molecules. In this state the SS cells are insufficiently flexible to navigate the channels of the tortuous tumor vasculature.
The SS and erythroleukemia cells of the claimed invention differ from other therapies in the field in that they are natural products ideally suited as carriers of tumoricidal agents specifically into tumor cells. They are abundantly available from the large pool of SS patients worldwide and do not require culture conditions for long term maintenance. The native SS erythroblasts and erythroleukemia cells target microvasculature of virtually all tumors without relying on the presence of antibodies or specific signaling molecules. They do not induce the immunosuppression of chemotherapy or the acute toxicity of various toxins. With conventional ABO blood typing SS erythrocytes and erythroleukemia cells can be used as safely in humans as a blood transfusion requiring only one tenth the volume of a conventional unit of blood. Moreover, SS cells do not induce major histoincompatibily-related reactions associated with the use of allogeneic leukocytes. Nor do SS erythroid progenitor cells since they exhibit minimal expression of MHC I and II molecules compared to mature leukocytes and platelets.